A long noncoding RNA at the cortex locus controls adaptive coloration in butterflies.

Evolutionary variation in the wing pigmentation of butterflies and moths offers striking examples of adaptation by crypsis and mimicry. The cortex locus has been independently mapped as the locus controlling color polymorphisms in 15 lepidopteran species, suggesting that it acts as a genomic hotspot for the diversification of wing patterns, but functional validation through protein-coding knockouts has proven difficult to obtain. Our study unveils the role of a long noncoding RNA (lncRNA) which we name ivory, transcribed from the cortex locus, in modulating color patterning in butterflies. Strikingly, ivory expression prefigures most melanic patterns during pupal development, suggesting an early developmental role in specifying scale identity. To test this, we generated CRISPR mosaic knock-outs in five nymphalid butterfly species and show that ivory mutagenesis yields transformations of dark pigmented scales into white or light-colored scales. Genotyping of Vanessa cardui germline mutants associates these phenotypes to small on-target deletions at the conserved first exon of ivory. In contrast, cortex germline mutant butterflies with confirmed null alleles lack any wing phenotype and exclude a color patterning role for this adjacent gene. Overall, these results show that a lncRNA gene acts as a master switch of color pattern specification and played key roles in the adaptive diversification of wing patterns in butterflies.

Cis-regulatory modes of Ultrabithorax inactivation in butterfly forewings.

Hox gene clusters encode transcription factors that drive regional specialization during animal development: for example the Hox factor Ubx is expressed in the insect metathoracic (T3) wing appendages and differentiates them from T2 mesothoracic identities. Hox transcriptional regulation requires silencing activities that prevent spurious activation and regulatory crosstalks in the wrong tissues, but this has seldom been studied in insects other than Drosophila, which shows a derived Hox dislocation into two genomic clusters that disjoined Antennapedia (Antp) and Ultrabithorax (Ubx). Here, we investigated how Ubx is restricted to the hindwing in butterflies, amidst a contiguous Hox cluster. By analysing Hi-C and ATAC-seq data in the butterfly Junonia coenia, we show that a Topologically Associated Domain (TAD) maintains a hindwing-enriched profile of chromatin opening around Ubx. This TAD is bordered by a Boundary Element (BE) that separates it from a region of joined wing activity around the Antp locus. CRISPR mutational perturbation of this BE releases ectopic Ubx expression in forewings, inducing homeotic clones with hindwing identities. Further mutational interrogation of two non-coding RNA encoding regions and one putative cis-regulatory module within the Ubx TAD cause rare homeotic transformations in both directions, indicating the presence of both activating and repressing chromatin features. We also describe a series of spontaneous forewing homeotic phenotypes obtained in Heliconius butterflies, and discuss their possible mutational basis. By leveraging the extensive wing specialization found in butterflies, our initial exploration of Ubx regulation demonstrates the existence of silencing and insulating sequences that prevent its spurious expression in forewings.

Frizzled2 receives WntA signaling during butterfly wing pattern formation.

Butterfly color patterns provide visible and biodiverse phenotypic readouts of the patterning processes. Although the secreted ligand WntA has been shown to instruct the color pattern formation in butterflies, its mode of reception remains elusive. Butterfly genomes encode four homologs of the Frizzled-family of Wnt receptors. Here, we show that CRISPR mosaic knockouts of frizzled2 (fz2) phenocopy the color pattern effects of WntA loss of function in multiple nymphalids. Whereas WntA mosaic clones result in intermediate patterns of reduced size, fz2 clones are cell-autonomous, consistent with a morphogen function. Shifts in expression of WntA and fz2 in WntA crispant pupae show that they are under positive and negative feedback, respectively. Fz1 is required for Wnt-independent planar cell polarity in the wing epithelium. Fz3 and Fz4 show phenotypes consistent with Wnt competitive-antagonist functions in vein formation (Fz3 and Fz4), wing margin specification (Fz3), and color patterning in the Discalis and Marginal Band Systems (Fz4). Overall, these data show that the WntA/Frizzled2 morphogen-receptor pair forms a signaling axis that instructs butterfly color patterning and shed light on the functional diversity of insect Frizzled receptors.

Genetics of yellow-orange color variation in a pair of sympatric sulphur butterflies.

Continuous color polymorphisms can serve as a tractable model for the genetic and developmental architecture of traits. Here we investigated continuous color variation in Colias eurytheme and Colias philodice, two species of sulphur butterflies that hybridize in sympatry. Using quantitative trait locus (QTL) analysis and high-throughput color quantification, we found two interacting large-effect loci affecting orange-to-yellow chromaticity. Knockouts of red Malpighian tubules (red), likely involved in endosomal maturation, result in depigmented wing scales. Additionally, the transcription factor bric-a-brac can act as a modulator of orange pigmentation. We also describe the QTL architecture of other continuously varying traits, together supporting a large-X effect model where the genetic control of species-defining traits is enriched on sex chromosomes. This study sheds light on the range of possible genetic architectures that can underpin a continuously varying trait and illustrates the power of using automated measurement to score phenotypes that are not always conspicuous to the human eye.

Evidence for a single, ancient origin of a genus-wide alternative life history strategy.

Understanding the evolutionary origins and factors maintaining alternative life history strategies (ALHS) within species is a major goal of evolutionary research. While alternative alleles causing discrete ALHS are expected to purge or fix over time, one-third of the ~90 species of Colias butterflies are polymorphic for a female-limited ALHS called Alba. Whether Alba arose once, evolved in parallel, or has been exchanged among taxa is currently unknown. Using comparative genome-wide association study (GWAS) and population genomic analyses, we placed the genetic basis of Alba in time-calibrated phylogenomic framework, revealing that Alba evolved once near the base of the genus and has been subsequently maintained via introgression and balancing selection. CRISPR-Cas9 mutagenesis was then used to verify a putative cis-regulatory region of Alba, which we identified using phylogenetic foot printing. We hypothesize that this cis-regulatory region acts as a modular enhancer for the induction of the Alba ALHS, which has likely facilitated its long evolutionary persistence.

High level of novelty under the hood of convergent evolution.

Little is known about the extent to which species use homologous regulatory architectures to achieve phenotypic convergence. By characterizing chromatin accessibility and gene expression in developing wing tissues, we compared the regulatory architecture of convergence between a pair of mimetic butterfly species. Although a handful of color pattern genes are known to be involved in their convergence, our data suggest that different mutational paths underlie the integration of these genes into wing pattern development. This is supported by a large fraction of accessible chromatin being exclusive to each species, including the de novo lineage-specific evolution of a modular optix enhancer. These findings may be explained by a high level of developmental drift and evolutionary contingency that occurs during the independent evolution of mimicry.

Erratum: Mapping and CRISPR homology-directed repair of a recessive white eye mutation in Plodia moths.

[This corrects the article DOI: 10.1016/j.isci.2022.103885.].

Mapping and CRISPR homology-directed repair of a recessive white eye mutation in Plodia moths.

The pantry moth Plodia interpunctella is a worldwide pest of stored food products and a promising laboratory model system for lepidopteran functional genomics. Here we describe efficient methods for precise genome editing in this insect. A spontaneous recessive white-eyed phenotype maps to a frameshift deletion (c.737delC) in the white gene. CRISPR NHEJ mutagenesis of white replicates this phenotype with high rates of somatic biallelic knockout. G0 individuals with mutant clones on both eyes produced 100% mutant progeny, making white an ideal marker for co-conversion when targeting other genes. CRISPR HDR experiments corrected c.737delC and reverted white eyes to a pigmented state in 37% of G0 mosaic adults. These repaired alleles showed practical rates of germline transmission in backcrosses, demonstrating the potential of the technique for precise genome editing. Plodia offers a promising avenue for research in this taxon because of its lab-ready features, egg injectability, and editability.

Knockdowns of red Malphigian tubules reveal pigmentation roles in the milkweed bug.

Classical Drosophila eye color mutations have unearthed a toolkit of genes that have permitted candidate gene studies of the outstanding diversity of coloration patterns in other insects. The gene underlying the eye color phenotypes of the red Malphigian tubules (red) fly mutant was mapped to a LysM domain gene of unknown molecular function. Here, we used RNAi to test the role of a red ortholog in the pigmentation of the milkweed bug Oncopeltus fasciatus, and contrast its effect with the ommochrome biosynthetic pathway gene vermilion (ver). Pigmentation was reduced in the cuticle of embryonic legs and first instar abdomens following parental RNAi against red, but not against ver, likely reflecting an effect on pterin biogenesis. Nymphal RNAi of red and ver both resulted in adult eye depigmentation, consistent with an effect on ommochrome content. These results suggest red loss-of-function impacts biochemically distinct types of pigments, and we discuss its putative role in the biogenesis of lysosome-related organelles such as ommochromasomes and pterinosomes.

A genetic switch for male UV iridescence in an incipient species pair of sulphur butterflies.

Mating cues evolve rapidly and can contribute to species formation and maintenance. However, little is known about how sexual signals diverge and how this variation integrates with other barrier loci to shape the genomic landscape of reproductive isolation. Here, we elucidate the genetic basis of ultraviolet (UV) iridescence, a courtship signal that differentiates the males of Colias eurytheme butterflies from a sister species, allowing females to avoid costly heterospecific matings. Anthropogenic range expansion of the two incipient species established a large zone of secondary contact across the eastern United States with strong signatures of genomic admixtures spanning all autosomes. In contrast, Z chromosomes are highly differentiated between the two species, supporting a disproportionate role of sex chromosomes in speciation known as the large-X (or large-Z) effect. Within this chromosome-wide reproductive barrier, linkage mapping indicates that cis-regulatory variation of bric a brac (bab) underlies the male UV-iridescence polymorphism between the two species. Bab is expressed in all non-UV scales, and butterflies of either species or sex acquire widespread ectopic iridescence following its CRISPR knockout, demonstrating that Bab functions as a suppressor of UV-scale differentiation that potentiates mating cue divergence. These results highlight how a genetic switch can regulate a premating signal and integrate with other reproductive barriers during intermediate phases of speciation.

A large deletion at the cortex locus eliminates butterfly wing patterning.

As the genetic basis of natural and domesticated variation has been described in recent years, a number of hotspot genes have been repeatedly identified as the targets of selection, Heliconius butterflies display a spectacular diversity of pattern variants in the wild and the genetic basis of these patterns has been well-described. Here, we sought to identify the mechanism behind an unusual pattern variant that is instead found in captivity, the ivory mutant, in which all scales on both the wings and body become white or yellow. Using a combination of autozygosity mapping and coverage analysis from 37 captive individuals, we identify a 78-kb deletion at the cortex wing patterning locus, a gene which has been associated with wing pattern evolution in H. melpomene and 10 divergent lepidopteran species. This deletion is undetected among 458 wild Heliconius genomes samples, and its dosage explains both homozygous and heterozygous ivory phenotypes found in captivity. The deletion spans a large 5' region of the cortex gene that includes a facultative 5'UTR exon detected in larval wing disk transcriptomes. CRISPR mutagenesis of this exon replicates the wing phenotypes from coding knock-outs of cortex, consistent with a functional role of ivory-deleted elements in establishing scale color fate. Population demographics reveal that the stock giving rise to the ivory mutant has a mixed origin from across the wild range of H. melpomene, and supports a scenario where the ivory mutation occurred after the introduction of cortex haplotypes from Ecuador. Homozygotes for the ivory deletion are inviable while heterozygotes are the targets of artificial selection, joining 40 other examples of allelic variants that provide heterozygous advantage in animal populations under artificial selection by fanciers and breeders. Finally, our results highlight the promise of autozygosity and association mapping for identifying the genetic basis of aberrant mutations in captive insect populations.

Cortex cis-regulatory switches establish scale colour identity and pattern diversity in Heliconius.

In Heliconius butterflies, wing colour pattern diversity and scale types are controlled by a few genes of large effect that regulate colour pattern switches between morphs and species across a large mimetic radiation. One of these genes, cortex, has been repeatedly associated with colour pattern evolution in butterflies. Here we carried out CRISPR knockouts in multiple Heliconius species and show that cortex is a major determinant of scale cell identity. Chromatin accessibility profiling and introgression scans identified cis-regulatory regions associated with discrete phenotypic switches. CRISPR perturbation of these regions in black hindwing genotypes recreated a yellow bar, revealing their spatially limited activity. In the H. melpomene/timareta lineage, the candidate CRE from yellow-barred phenotype morphs is interrupted by a transposable element, suggesting that cis-regulatory structural variation underlies these mimetic adaptations. Our work shows that cortex functionally controls scale colour fate and that its cis-regulatory regions control a phenotypic switch in a modular and pattern-specific fashion.

Heliconius butterflies have bright patterns on their wings that tell potential predators that they are toxic. As a result, predators learn to avoid eating them. Over time, unrelated species of butterflies have evolved similar patterns to avoid predation through a process known as Müllerian mimicry. Worldwide, there are over 180,000 species of butterflies and moths, most of which have different wing patterns. How do genes create this pattern diversity? And do butterflies use similar genes to create similar wing patterns? One of the genes involved in creating wing patterns is called cortex. This gene has a large region of DNA around it that does not code for proteins, but instead, controls whether cortex is on or off in different parts of the wing. Changes in this non-coding region can act like switches, turning regions of the wing into different colours and creating complex patterns, but it is unclear how these switches have evolved. Butterfly wings get their colour from tiny structures called scales, which each have their own unique set of pigments. In Heliconius butterflies, there are three types of scales: yellow/white scales, black scales, and red/orange/brown scales. Livraghi et al. used a DNA editing technique called CRISPR to find out whether the cortex gene affects scale type. First, Livraghi et al. confirmed that deleting cortex turned black and red scales yellow. Next, they used the same technique to manipulate the non-coding DNA around the cortex gene to see the effect on the wing pattern. This manipulation turned a black-winged butterfly into a butterfly with a yellow wing band, a pattern that occurs naturally in Heliconius butterflies. The next step was to find the mutation responsible for the appearance of yellow wing bands in nature. It turns out that a bit of extra genetic code, derived from so-called 'jumping genes', had inserted itself into the non-coding DNA around the cortex gene, 'flipping' the switch and leading to the appearance of the yellow scales. Genetic information contains the instructions to generate shape and form in most organisms. These instructions evolve over millions of years, creating everything from bacteria to blue whales. Butterfly wings are visual evidence of evolution, but the way their genes create new patterns isn't specific to butterflies. Understanding wing patterns can help researchers to learn how genetic switches control diversity across other species too.

Porcupine/Wntless-dependent trafficking of the conserved WntA ligand in butterflies.

Wnt ligands are key signaling molecules in animals, but little is known about the evolutionary dynamics and mode of action of the WntA orthologs, which are not present in the vertebrates or in Drosophila. Here we show that the WntA subfamily evolved at the base of the Bilateria + Cnidaria clade, and conserved the thumb region and Ser209 acylation site present in most other Wnts, suggesting WntA requires the core Wnt secretory pathway. WntA proteins are distinguishable from other Wnts by a synapomorphic Iso/Val/Ala216 amino-acid residue that replaces the otherwise ubiquitous Thr216 position. WntA embryonic expression is conserved between beetles and butterflies, suggesting functionality, but the WntA gene was lost three times within arthropods, in podoplean copepods, in the cyclorrhaphan fly radiation, and in ensiferan crickets and katydids. Finally, CRISPR mosaic knockouts (KOs) of porcupine and wntless phenocopied the pattern-specific effects of WntA KOs in the wings of Vanessa cardui butterflies. These results highlight the molecular conservation of the WntA protein across invertebrates, and imply it functions as a typical Wnt ligand that is acylated and secreted through the Porcupine/Wntless secretory pathway.

Deep Convergence, Shared Ancestry, and Evolutionary Novelty in the Genetic Architecture of Heliconius Mimicry.

Convergent evolution can occur through different genetic mechanisms in different species. It is now clear that convergence at the genetic level is also widespread, and can be caused by either (i) parallel genetic evolution, where independently evolved convergent mutations arise in different populations or species, or (ii) collateral evolution in which shared ancestry results from either ancestral polymorphism or introgression among taxa. The adaptive radiation of Heliconius butterflies shows color pattern variation within species, as well as mimetic convergence between species. Using comparisons from across multiple hybrid zones, we use signals of shared ancestry to identify and refine multiple putative regulatory elements in Heliconius melpomene and its comimics, Heliconius elevatus and Heliconius besckei, around three known major color patterning genes: optix, WntA, and cortex While we find that convergence between H. melpomene and H. elevatus is caused by a complex history of collateral evolution via introgression in the Amazon, convergence between these species in the Guianas appears to have evolved independently. Thus, we find adaptive convergent genetic evolution to be a key driver of regulatory changes that lead to rapid phenotypic changes. Furthermore, we uncover evidence of parallel genetic evolution at some loci around optix and WntA in H. melpomene and its distant comimic Heliconius erato Ultimately, we show that all three of convergence, conservation, and novelty underlie the modular architecture of Heliconius color pattern mimicry.

Interplay between Developmental Flexibility and Determinism in the Evolution of Mimetic Heliconius Wing Patterns.

To what extent can we predict how evolution occurs? Do genetic architectures and developmental processes canalize the evolution of similar outcomes in a predictable manner? Or do historical contingencies impose alternative pathways to answer the same challenge? Examples of Müllerian mimicry between distantly related butterfly species provide natural replicates of evolution, allowing us to test whether identical wing patterns followed parallel or novel trajectories. Here, we explore the role that the signaling ligand WntA plays in generating mimetic wing patterns in Heliconius butterflies, a group with extraordinary mimicry-related wing pattern diversity. The radiation is relatively young, and numerous cases of wing pattern mimicry have evolved within the last 2.5-4.5 Ma. WntA is an important target of natural selection and is one of four major effect loci that underlie much of the pattern variation in the group. We used CRISPR/Cas9 targeted mutagenesis to generate WntA-deficient wings in 12 species and a further 10 intraspecific variants, including three co-mimetic pairs. In all tested butterflies, WntA knockouts affect pattern broadly and cause a shift among every possible scale cell type. Interestingly, the co-mimics lacking WntA were very different, suggesting that the gene networks that pattern a wing have diverged considerably among different lineages. Thus, although natural selection channeled phenotypic convergence, divergent developmental contexts between the two major Heliconius lineages opened different developmental routes to evolve resemblance. Consequently, even under very deterministic evolutionary scenarios, our results underscore a surprising unpredictability in the developmental paths underlying convergence in a recent radiation.

Conservation and flexibility in the gene regulatory landscape of heliconiine butterfly wings.

BACKGROUND: Many traits evolve by cis-regulatory modification, by which changes to noncoding sequences affect the binding affinity for available transcription factors and thus modify the expression profile of genes. Multiple examples of cis-regulatory evolution have been described at pattern switch genes responsible for butterfly wing pattern polymorphism, including in the diverse neotropical genus Heliconius, but the identities of the factors that can regulate these switch genes have not been identified. RESULTS: We investigated the spatial transcriptomic landscape across the wings of three closely related butterfly species, two of which have a convergently evolved co-mimetic pattern and the other having a divergent pattern. We identified candidate factors for regulating the expression of wing patterning genes, including transcription factors with a conserved expression profile in all three species, and others, including both transcription factors and Wnt pathway genes, with markedly different profiles in each of the three species. We verified the conserved expression profile of the transcription factor homothorax by immunofluorescence and showed that its expression profile strongly correlates with that of the selector gene optix in butterflies with the Amazonian forewing pattern element 'dennis.' CONCLUSION: Here we show that, in addition to factors with conserved expression profiles like homothorax, there are also a variety of transcription factors and signaling pathway components that appear to vary in their expression profiles between closely related butterfly species, highlighting the importance of genome-wide regulatory evolution between species.

Sub-micrometer insights into the cytoskeletal dynamics and ultrastructural diversity of butterfly wing scales.

BACKGROUND: The color patterns that adorn lepidopteran wings are ideal for studying cell type diversity using a phenomics approach. Color patterns are made of chitinous scales that are each the product of a single precursor cell, offering a 2D system where phenotypic diversity can be studied cell by cell, both within and between species. Those scales reveal complex ultrastructures in the sub-micrometer range that are often connected to a photonic function, including iridescent blues and greens, highly reflective whites, or light-trapping blacks. RESULTS: We found that during scale development, Fascin immunostainings reveal punctate distributions consistent with a role in the control of actin patterning. We quantified the cytoskeleton regularity as well as its relationship to chitin deposition sites, and confirmed a role in the patterning of the ultrastructures of the adults scales. Then, in an attempt to characterize the range and variation in lepidopteran scale ultrastructures, we devised a high-throughput method to quickly derive multiple morphological measurements from fluorescence images and scanning electron micrographs. We imaged a multicolor eyespot element from the butterfly Vanessa cardui (V. cardui), taking approximately 200 000 individual measurements from 1161 scales. Principal component analyses revealed that scale structural features cluster by color type, and detected the divergence of non-reflective scales characterized by tighter cross-rib distances and increased orderedness. CONCLUSION: We developed descriptive methods that advance the potential of butterfly wing scales as a model system for studying how a single cell type can differentiate into a multifaceted spectrum of complex morphologies. Our data suggest that specific color scales undergo a tight regulation of their ultrastructures, and that this involves cytoskeletal dynamics during scale growth.

Complex modular architecture around a simple toolkit of wing pattern genes.

Identifying the genomic changes that control morphological variation and understanding how they generate diversity is a major goal of evolutionary biology. In Heliconius butterflies, a small number of genes control the development of diverse wing color patterns. Here, we used full genome sequencing of individuals across the Heliconius erato radiation and closely related species to characterize genomic variation associated with wing pattern diversity. We show that variation around color pattern genes is highly modular, with narrow genomic intervals associated with specific differences in color and pattern. This modular architecture explains the diversity of color patterns and provides a flexible mechanism for rapid morphological diversification.

Waiting in the wings: what can we learn about gene co-option from the diversification of butterfly wing patterns?

A major challenge is to understand how conserved gene regulatory networks control the wonderful diversity of form that we see among animals and plants. Butterfly wing patterns are an excellent example of this diversity. Butterfly wings form as imaginal discs in the caterpillar and are constructed by a gene regulatory network, much of which is conserved across the holometabolous insects. Recent work in Heliconius butterflies takes advantage of genomic approaches and offers insights into how the diversification of wing patterns is overlaid onto this conserved network. WntA is a patterning morphogen that alters spatial information in the wing. Optix is a transcription factor that acts later in development to paint specific wing regions red. Both of these loci fit the paradigm of conserved protein-coding loci with diverse regulatory elements and developmental roles that have taken on novel derived functions in patterning wings. These discoveries offer insights into the 'Nymphalid Ground Plan', which offers a unifying hypothesis for pattern formation across nymphalid butterflies. These loci also represent 'hotspots' for morphological change that have been targeted repeatedly during evolution. Both convergent and divergent evolution of a great diversity of patterns is controlled by complex alleles at just a few genes. We suggest that evolutionary change has become focused on one or a few genetic loci for two reasons. First, pre-existing complex cis-regulatory loci that already interact with potentially relevant transcription factors are more likely to acquire novel functions in wing patterning. Second, the shape of wing regulatory networks may constrain evolutionary change to one or a few loci. Overall, genomic approaches that have identified wing patterning loci in these butterflies offer broad insight into how gene regulatory networks evolve to produce diversity.This article is part of the themed issue 'Evo-devo in the genomics era, and the origins of morphological diversity'.